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Development and optimization of two-dimensional-electrophoresis protocol ofLeptospirillum ferriphilum

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Leptospirillum ferriphilum is important in bioleaching, in which process it is often under heavy stresses of heavy metal ions and high oxidation reduction potential (ORP). Two-dimensional-electrophoresis (2-DE) and comparative proteomic analysis are useful to investigate the responses ofL. ferriphilum to environmental stresses. But, 2-DE analysis forL. ferriphilum is not successful as the samples ofL. ferriphilum contain low protein concentration, complex composition, high salt concentration, and many other interfering components, which make it difficult for 2-DE analysis. In this research, optimizations on the sample preparation and purification methods, sample volume, sample loading methods for isoelectric focusing (IEF), and gel visualization methods were made. More than 629 Coomassie stained spots in single gel were obtained. The image quality and protein concentration in most of the spots met the requirements for both differential spots analysis and mass-spectrum analysis. The 2-DE protocol forL. ferriphilum was successfully developed for the first time.

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  1. Candiano G., Bruschi M., Musante L., Santucci L., Ghiggeri G.M., Carnemolla B., Orecchia P., Zardi L., Righetti P.G. (2004). Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis, 25: 1327–1333.

  2. Coram N.J., Rawlings D.E. (2002). Molecular relationship between two groups of the genusLeptospirillum and the finding thatLeptospirillum ferriphilum sp. nov. dominates South African commercial biooxidation tanks that operate at 40 degrees C. Appl. Environ. Microbiol., 68: 838–845.

  3. Liu J., Xie X., Xiao S., Wang X., Zhao W., Tian Z. (2007). Isolation ofLeptospirillum ferriphilum by single-layered solid medium. J. Cent. South Univ. Technol., 4: 467–473.

  4. Liu Y., Liu X., Tian K., Qi E., Yan W. (2003). Growth and morphology ofLeptospirillum ferrooxidans on solid medium. Microbiology, 30: 70–72 (in Chinese).

  5. Liu Y., Liu X., Zheng L., Lin J., Yan W. (2004). Isolation and characterization of a vibrioid-shaped iron-oxidizing bacterium. J. Shandong Univ., 39: 116–124 (in Chinese).

  6. Ram R.J., VerBerkmoes N.C., Thelen M.P., Tyson G.W., Baker B.J., Blake R.C., Shah M., Hettich R.L., Banfield J.F. (2005). Community proteomicsofa natural microbial biofilm. Science, 308: 1915–1920.

  7. Rawlings D.E., Tributsch H., Hansford G.S. (1999). Reasons why ‘Leptospirillum’-like species rather thanThiobacillus ferrooxidans are the dominant iron-oxidizing bacteria in many commercial processes for the biooxidation of pyrite and related ores. Microbiology, 145: 5–13.

  8. Rosenkrands I., Weldingh K., Jacobsen S. (2000). Mapping and identification of mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection. Electrophoresis, 21: 935–948.

  9. Sun B., Fan L., Li L., Zhu W., Lu G. (2006). Establishment of the 2-D synthetic map of total protein of normal human spermatozoa enriched with low abundance protein. National J. of Andrology, 12: 295–299 (in Chinese).

  10. Tuffin I.M., Hector S.B., Deane S.M., Rawlings D.E. (2006). Resistance determinants of a highly arsenic-resistant strain ofLeptospirillum ferriphilum isolated from a commercial biooxidation tank. Appl. Environ. Microbiol., 72: 2247–2253.

  11. Tyson G.W., Chapman J., Hugenholtz P., Allen E.E., Ram R.J., Richardson P.M., Solovyev V.V., Rubin E.M., Rokhsar D.S., Banfield J.F. (2004). Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature, 428: 25–26.

  12. Wang J. (2002). Preparation for Protein’s Separation, Protein Technology Manual, Science Press, Beijing (In Chinese).

  13. Wang S.B., Hu Q., Sommerfeld M., Chen F. (2003). An optimized protocol for isolation of soluble proteins from microalgae for two-dimensional gel electrophoresis analysis. J. Appl. Phycol., 15: 485–496.

  14. Wang M., Peng Z., Li C., Li F., Liu C., Xia G. (2008). Proteomic analysis on a high salt tolerance introgression strain ofTriticum aestivum/Thinopyrum ponticum. Proteomics, 8:1470–1489.

  15. Yin P., Wang Y.H., Zhang S.L., Chu J., Zhuang Y.P., Wang M.L., Zhou J. (2008). Isolation of soluble proteins from an industrial strainStreptomyces avermitilis in complex culture medium for two-dimensional gel electrophoresis. J. Microbiol Methods, 73: 105–110.

  16. Zhang X., Xiao Z., Chen Z., Li C., Li J., Yanhui Y., Yang F., Yang Y., Oyang Y (2006). Comparative proteomics analysis of the proteins associated with laryngeal carcinoma-related gene 1. Laryngoscope, 116: 224–230.

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Correspondence to Jianqun Lin.

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Li, B., Lin, J., Lin, J. et al. Development and optimization of two-dimensional-electrophoresis protocol ofLeptospirillum ferriphilum . Ann. Microbiol. 59, 833–838 (2009).

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  • Leptospirillum ferriphilum
  • two-dimensional-electrophoresis
  • proteomics
  • bioleaching